Daniel L. Barber

Restoring function in exhausted CD8 T cells during chronic viral infection.

Functional impairment of antigen-specific T cells is a defining characteristic of many chronic infections, but the underlying mechanisms of T-cell dysfunction are not well understood. To address this question, we analysed genes expressed in functionally impaired virus-specific CD8 T cells present in mice chronically infected with lymphocytic choriomeningitis virus (LCMV), and compared these with the gene profile of functional memory CD8 T cells. Here we report that PD-1 (programmed death 1; also known as Pdcd1) was selectively upregulated by the exhausted T cells, and that in vivo administration of antibodies that blocked the interaction of this inhibitory receptor with its ligand, PD-L1 (also known as B7-H1), enhanced T-cell responses. Notably, we found that even in persistently infected mice that were lacking CD4 T-cell help, blockade of the PD-1/PD-L1 inhibitory pathway had a beneficial effect on the ‘helpless’ CD8 T cells, restoring their ability to undergo proliferation, secrete cytokines, kill infected cells and decrease viral load. Blockade of the CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) inhibitory pathway had no effect on either T-cell function or viral control. These studies identify a specific mechanism of T-cell exhaustion and define a potentially effective immunological strategy for the treatment of chronic viral infections.

Cutting Edge: Rapid In Vivo Killing by Memory CD8 T Cells

In this study, we examined the cytotoxic activity of effector and memory CD8 T cells in vivo. At the peak of the CTL response following an acute lymphocytic choriomeningitis virus infection, effector CD8 T cells exhibited extremely rapid killing and started to eliminate adoptively transferred target cells within 15 min by a perforin-dependent mechanism. Although resting memory CD8 T cells are poorly cytolytic by in vitro 51Cr release assays, there was rapid elimination (within 1–4 h) of target cells after transfer into immune mice, and both CD62Lhigh and CD62Llow memory CD8 T cells were able to kill rapidly in vivo. Strikingly, when directly compared on a per cell basis, memory CD8 T cells were only slightly slower than effector cells in eliminating target cells. These data indicate that virus specific memory CD8 T cells can rapidly acquire cytotoxic function upon re-exposure to Ag and are much more efficient killers in vivo than previously appreciated.

Cutting Edge: Caspase-1 Independent IL-1β Production Is Critical for Host Resistance to Mycobacterium tuberculosis and Does Not Require TLR Signaling In Vivo

To investigate the respective contributions of TLR versus IL-1R mediated signals in MyD88 dependent control of Mycobacterium tuberculosis, we compared the outcome of M. tuberculosis infection in MyD88, TRIF/MyD88, IL-1R1, and IL-1β–deficient mice. All four strains displayed acute mortality with highly increased pulmonary bacterial burden suggesting a major role for IL-1β signaling in determining the MyD88 dependent phenotype. Unexpectedly, the infected MyD88 and TRIF/MyD88-deficient mice, rather than being defective in IL-1β expression, displayed increased cytokine levels relative to wild-type animals. Similarly, infected mice deficient in caspase-1 and ASC, which have critical functions in inflammasome-mediated IL-1β maturation, showed unimpaired IL-1β production and importantly, were considerably less susceptible to infection than IL-1β deficient mice. Together our findings reveal a major role for IL-1β in host resistance to M. tuberculosis and indicate that during this infection the cytokine can be generated by a mechanism that does not require TLR signaling or caspase-1.

Host-directed therapy of tuberculosis based on interleukin-1 and type I interferon crosstalk

Tuberculosis remains second only to HIV/AIDS as the leading cause of mortality worldwide due to a single infectious agent. Despite chemotherapy, the global tuberculosis epidemic has intensified because of HIV co-infection, the lack of an effective vaccine and the emergence of multi-drug-resistant bacteria. Alternative host-directed strategies could be exploited to improve treatment efficacy and outcome, contain drug-resistant strains and reduce disease severity and mortality. The innate inflammatory response elicited by Mycobacterium tuberculosis (Mtb) represents a logical host target. Here we demonstrate that interleukin-1 (IL-1) confers host resistance through the induction of eicosanoids that limit excessive type I interferon (IFN) production and foster bacterial containment. We further show that, in infected mice and patients, reduced IL-1 responses and/or excessive type I IFN induction are linked to an eicosanoid imbalance associated with disease exacerbation. Host-directed immunotherapy with clinically approved drugs that augment prostaglandin E2 levels in these settings prevented acute mortality of Mtb-infected mice. Thus, IL-1 and type I IFNs represent two major counter-regulatory classes of inflammatory cytokines that control the outcome of Mtb infection and are functionally linked via eicosanoids. Our findings establish proof of concept for host-directed treatment strategies that manipulate the host eicosanoid network and represent feasible alternatives to conventional chemotherapy.

Dynamic T cell migration program provides resident memory within intestinal epithelium

Migration to intestinal mucosa putatively depends on local activation because gastrointestinal lymphoid tissue induces expression of intestinal homing molecules, whereas skin-draining lymph nodes do not. This paradigm is difficult to reconcile with reports of intestinal T cell responses after alternative routes of immunization. We reconcile this discrepancy by demonstrating that activation within spleen results in intermediate induction of homing potential to the intestinal mucosa. We further demonstrate that memory T cells within small intestine epithelium do not routinely recirculate with memory T cells in other tissues, and we provide evidence that homing is similarly dynamic in humans after subcutaneous live yellow fever vaccine immunization. These data explain why systemic immunization routes induce local cell-mediated immunity within the intestine and indicate that this tissue must be seeded with memory T cell precursors shortly after activation.

Cutting Edge: Gut Microenvironment Promotes Differentiation of a Unique Memory CD8 T Cell Population

Whether tissue microenvironment influences memory CD8 T cell differentiation is unclear. We demonstrate that virus-specific intraepithelial lymphocytes in gut resemble neither central nor effector memory CD8 T cells isolated from spleen or blood. This unique phenotype arises in situ within the gut, suggesting that anatomic location plays an inductive role in the memory differentiation program. In support of this hypothesis, memory CD8 T cells changed phenotype upon change in location. After transfer and in vivo restimulation, gut or spleen memory cells proliferated, disseminated into spleen and gut, and adopted the memory T cell phenotype characteristic of their new environment. Our data suggests that anatomic location directly impacts the memory T cell differentiation program.

Innate and adaptive interferons suppress IL-1α and IL-1β production by distinct pulmonary myeloid subsets during Mycobacterium tuberculosis infection.

Interleukin-1 (IL-1) receptor signaling is necessary for control of Mycobacterium tuberculosis (Mtb) infection, yet the role of its two ligands, IL-1α and IL-1β, and their regulation in vivo are poorly understood. Here, we showed that both IL-1α and IL-1β are critically required for host resistance and identified two multifunctional inflammatory monocyte-macrophage and DC populations that coexpressed both IL-1 species at the single-cell level in lungs of Mtb-infected mice. Moreover, we demonstrated that interferons (IFNs) played important roles in regulating IL-1 production by these cells in vivo. Type I interferons inhibited IL-1 production by both subsets whereas CD4(+) T cell-derived IFN-γ selectively suppressed monocyte-macrophages. These data provide a cellular basis for both the anti-inflammatory effects of IFNs and probacterial functions of type I IFNs during Mtb infection and reveal differential regulation of IL-1 production by distinct cell populations as an additional layer of complexity in the activity of IL-1 in vivo.

Enhancing therapeutic vaccination by blocking PD-1–mediated inhibitory signals during chronic infection

Therapeutic vaccination is a potentially promising strategy to enhance T cell immunity and viral control in chronically infected individuals. However, therapeutic vaccination approaches have fallen short of expectations, and effective boosting of antiviral T cell responses has not always been observed. One of the principal reasons for the limited success of therapeutic vaccination is that virus-specific T cells become functionally exhausted during chronic infections. We now provide a novel strategy for enhancing the efficacy of therapeutic vaccines. In this study, we show that blocking programmed death (PD)-1/PD-L1 inhibitory signals on exhausted CD8+ T cells, in combination with therapeutic vaccination, synergistically enhances functional CD8+ T cell responses and improves viral control in mice chronically infected with lymphocytic choriomeningitis virus. This combinatorial therapeutic vaccination was effective even in the absence of CD4+ T cell help. Thus, our study defines a potent new approach to augment the efficacy of therapeutic vaccination by blocking negative signals. Such an approach may have broad applications in developing treatment strategies for chronic infections in general, and perhaps also for tumors.

Intravascular staining for discrimination of vascular and tissue leukocytes

Characterization of the cellular participants in tissue immune responses is crucial to understanding infection, cancer, autoimmunity, allergy, graft rejection and other immunological processes. Previous reports indicate that leukocytes in lung vasculature fail to be completely removed by perfusion. Several studies suggest that intravascular staining may discriminate between tissue-localized and blood-borne cells in the mouse lung. Here we outline a protocol for the validation and use of intravascular staining to define innate and adaptive immune cells in mice. We demonstrate application of this protocol to leukocyte analyses in many tissues and we describe its use in the contexts of lymphocytic choriomeningitis virus and Mycobacterium tuberculosis infections or solid tumors. Intravascular staining and organ isolation usually takes 5–30 min per mouse, with additional time required for any subsequent leukocyte isolation, staining and analysis. In summary, this simple protocol should help enable interpretable analyses of tissue immune responses.

Continuous recruitment of naive T cells contributes to heterogeneity of antiviral CD8 T cells during persistent infection

Numerous microbes establish persistent infections, accompanied by antigen-specific CD8 T cell activation. Pathogen-specific T cells in chronically infected hosts are often phenotypically and functionally variable, as well as distinct from T cells responding to nonpersistent infections; this phenotypic heterogeneity has been attributed to an ongoing reencounter with antigen. Paradoxically, maintenance of memory CD8 T cells to acutely resolved infections is antigen independent, whereas there is a dependence on antigen for T cell survival in chronically infected hosts. Using two chronic viral infections, we demonstrate that new naive antigen-specific CD8 T cells are primed after the acute phase of infection. These newly recruited T cells are phenotypically distinct from those primed earlier. Long-lived antiviral CD8 T cells are defective in self-renewal, and lack of thymic output results in the decline of virus-specific CD8 T cells, indicating that newly generated T cells preserve antiviral CD8 T cell populations during chronic infection. These findings reveal a novel role for antigen in maintaining virus-specific CD8 T cells during persistent infection and provide insight toward understanding T cell differentiation in chronic infection.